Method of preparing substantially cell-free somatic antigens



United States Patent 3,408,440 METHOD OF PREPARING SUBSTANTIALLY CELL-FREE SOMATIC ANTIGENS Jack G. Voss, Wyoming, Ohio, assignor to The Procter glgamble Company, Cincinnati, Ohio, a corporation of No Drawing. Filed Sept. 26, 1966, Ser. No. 581,773

11 Claims. (Cl. 424-12) This invention relates to a method of preparing substantially cell-free somatic antigens. These antigens have analytical utility in that they act in precipitin tests to detect specific antibodies.

In a precipitin test, precipitation takes place when an antigen containing soluble constituents, such as a substantially cell-free somatic antigen prepared by the method of the present invention, and a serum containing specific antibody are mixed in appropriate proportions under specified test conditions, for example, as described hereinafter. This precipitation identifies the presence of the specific antibody.

Substantially cell-free somatic antigens for use as antibody-identifying agents in precipitin tests are known to have been prepared in the prior art by extraction from whole cells by the use of either aqueous trichloroacetic acid solutions or phenol-water mixtures as extracting agents. The method of the present invention dilfers from these prior art methods in that it employs different and more eflicient extracting agents than those of the prior art.

It is an object of this invention to provide an improved method for extracting somatic antigens from Escherichia coli and Salmonella typhosa.

It is another object of this invention to provide a method for obtaining essentially cell-free somatic antigens which are useful as antibody-identifying agents in precipitin tests.

According to this invention, it has been found that substantially cell-free somatic antigens can be prepared by a method comprising the steps of .(1) washing intact cells of E. coli or'S. typhosa in an aqueous solution of specified antigen-extracting agents under certain essential conditions as described hereinafter whereby somatic antigens are extracted from the cell walls; and (2) separating the extracted antigens from the remaining whole cells and cell walls.

The particular extracting agent for use in the first step of this method, i.e., in the washing and extraction step, depends upon whether the cells to be washed are E. coli or S. typhosa. If E. coli cells are to be washed, the extracting agents for use herein are organic compounds which form organic cations in aqueous solution, selected from the group consisting of:

(a) amines having the formula R 1L- wherein R is an alkyl group containing from about 8 to about 18 carbon atoms and having as substituents from to about 1 amine group having the formula R1 I I and from 0 to about 2 halogen atoms and wherein each R group is selected from the group consisting of hydrogen, alkyl groups containing from 1 to about 3 carbon atoms and hydroxy alkyl groups containing from 1 to about 3 carbon atoms;

ice

2 (b) quaternary ammonium compounds having the formula R2 [RI I -R x wherein R has the definition hereinbefore given and each R group is selected from the group consisting of alkyl groups containing from 1 to about 3 carbon atoms, mono halogen substituted alkyl groups containing from 1 to about 3 carbon atoms, benzyl groups and hydroxy alkyl groups containing from 1 to about 3 carbon atoms, and wherein X is selected from the group consisting of iodide, bromide, methosulfate, ethosulfate and chloride anions;

(c) N-alkyl piperidines wherein the alkyl groups contains from about 8 to about 18 carbon atoms;

(d) alkyl pyridinium halides wherein the alkyl group contains from about 8 to about 18 carbon atoms and the halide is selected from the group consisting of bromide, chloride and iodide;

(e) N-alkyl alkylene diamines wherein the alkyl group contains from about 8 to about 18 carbon atoms and the alkylene group contains from about 2 to about 4 carbon atoms;

(f) sulfonium and sulfoxonium compounds having the formula wherein R is a hydrocarbon group containing from about 8 to about 18 carbon atoms, wherein each R group is an alkyl group containing from 1 to about 3 carbon atoms, wherein X has the definition hereinbefore given (e.g., selected from the group consisting of iodide, bromide, chloride, methosulfate and ethosulfate anions), and Y is selected from the group consisting'of groups; (g) 1 alkyl 2 imino imidazolidines wherein the alkyl group contains from about 8 to about 18 carbon wherein R is a hydrocarbon group containing from 1 to about 24 carbon atoms, wherein each R group is a hydrocarbon group containing from 1 to about 4 carbon atoms, wherein X is selected from the group consisting of chlorine, iodine and bromine atoms, wherein L, m, and n are integers such that L is an integer from 0 to about 50, the sum of m and n is from about'2 to about 5, and the sum of m, n and the number of carbon atoms in R is greater than 12; and (i) mixtures thereof. If S. typhosa cells are to be washed instead of E. coli cells as above, these same extracting agents can be used, and additionally the sodium and potassium salts of ethylene- I a a s diaminetetraacetic acid (EDTA.) and mixtures of EDTA with the above extracting agents can be used.

The above extracting agents which form organic cations in aqueous solution are oftwo types. The first type is an amine e.g.,

which in aqueous solutionforms the cations R1 is RNll The above organic compounds (a), (c), (e) and (g) are of this type. The second type is a quaternized amine, sulfonium or sulfoxonium compound which already contains a cation which is associated with an anion (X or X and which dissociates in an aqueous solution to form the cation e9 (e.g., R-N-R Organic compounds (b), (d), (f) and (h) are of this type.

Amines suitable for use herein as extracting agents, compound (a) above, includes, for example, dimethyldodecylamine (DDA), dimethyloctylamine, decyldimethylamine, dimethyltetradecylamine, dimethylhexadecylamine, dimethyloctadecylamine, dodecylamine, dodecylethylamine, decylmethylpropylamine, decylhydroxymethylpropylamine.

Quaternary ammonium compounds suitable for use herein as extracting agents, compounds (b) above, includes, for example, dodecyltrimethylammonium,bromide (DTAB), tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium iodide, decyltrimethylammoni- I um bromide, C to C alkyl dimethylbenzyl ammonium chloride (Roccal), cetyldimethylbenzyl ammonium chloride, dimethyldodecylbenzyl ammonium bromide, dimethyltetradecylbenzyl ammonium iodide, chloromethyldiethyltetradecylammonium chloride, dodecyltriethanolammonium methosulfate, and tetradecylmethyldiethanolammonium ethosulfate.

Other compounds suitable for use herein as extracting agents which are examples of compounds (c), (d), (e), (f), (g), and (h) above, include N-dodecyl piperidine, cetyl pyridinium chloride, N-dodecyl-1,3-propylene diamine, dimethyldodecyl sulfonium iodide, dimethyldodecylsulfoxonium methosulfate, 1-dodecyl-2-imino imidazolidine, and polymerized epichlorohydrin (5 moles of monomer per molecule) quaternized with three moles of dimethyldodecylamine.

Preferred agents for extracting somatic antigens from E. coli cells include DDA, DTAB, and Roccal. Preferred agents for extracting somatic antigens from S. typhosa cells include DDA, DTAB, Roccal, and EDTA.

Except for the differences in the particular extracting agents described above, the same essential conditions can be employed in the first step of the present method for the washing of, and extraction of antigens from, either E. coli cells or S. typhosa cells. The essential conditions which must be employed in this first step include limits on cell concentration, extracting agent concentration, weight ratio of cells to extracting agent, pH, and temperature. The use of the above described extracting agents under such essential conditions as are hereinafter specified permits the extraction of cell=free antigens in accordance with this invention.

In the washing and extraction step, aqueous cell suspensions are prepared wherein the cell concentration ranges from about 5 micrograms to about 100 milligrams (mg) of cells per ml. of water. Cell weights specified herein are based on dry weight of cells and are measured by drying an aqueous cell suspension sample at C. for two hours and weighing the residue. Cell concentration is determined by dividing the weight of the residue by the volume of the initial suspension sample previous to drying. In practice concentrations in terms of dry weight are correlated with cell suspension optical density, and a cell suspension of specified concentration is prepared by diluting cells in an aqueous medium until the optical density which corresponds to the specified concentration is achieved. The extracting agent is mixed with the prepared aqueous cell suspension to form the aqueous extracting agent solution, in which the cells are washed in order to extract their somatic antigens, in an amount such that the concentration of the extracting agent in the solution ranges from about 1 p.p.m. to about 1000 p.p.m. The weight ratio of cells (dry weight as defined above) to extracting agent employed herein ranges from about 5:1 to about 100:1. The pH maintained during extraction ranges from about 4 to about 12. The temperature maintained during extraction ranges from about 5 C. to about C. All percentages and parts herein are by weight unless otherwise specified.

If cell concentrations less than about 5 micrograms/ml, extracting agent concentrations less than about 1 p.p.m., or weight ratios of cells to extracting agent less than about 5:1 are used herein, only a very small amount of somatic antigens is extracted, and these extracted antigens are ditficult to recover. If cell concentrations greater than about 100 mg./ml. or weight ratios of cells to extracting agent exceeding about 100:1 are employed herein, somatic antigens are not extracted from a large number of intact cells, and these cells are eventually disposed to waste. If extracting agent concentration exceeds about 1000 p.p.m., the excess extracting agent can contaminate the recovered product. The use of a pH outside the above described range interferes with antigen extraction. The lower limit for extraction temperature, i.e., about 5 C., is chosen so as to be slightly above the freezing point of the aqueous solution in which extraction is carried out. The use of a temperature of extraction exceeding about 150 C. can lead to the production of product without antigenic properties.

The time of washing and extraction is not particularly critical but preferably ranges from about 30 seconds to about 5 hours. At least 30 seconds are ordinarily required to physically carry out this washing and extraction step. There is no particular benefit achieved by carrying out the extraction step for more than about 5 hours.

Preferably, the cell concentration ranges from about 1 mg./ml. to about 20 mg./ml., the extracting agent concentration ranges from about 25 p.p.m. to about 500 p.p.m., the pH ranges from about 6 to about 9, and the temperature ranges from about 35 C. to about 40 C., especially 37 C. (98.6 F.). The most preferred time of washing and extraction ranges from about 5 minutes to about 20 minutes.

The present washing and extraction step is preferably carried out by suspending high concentrations of cells in water and dissolving the extracting agent in this suspension whereby the cells are washed with the extracting agent. For convenience in handling, the extracting agent is generally added to the cell suspension as a solution, for example, as a 2% solution; amine extracting agents, compounds (a), (c), (e), and (g) hereinbefore, are preferably dissolved in a lower alcohol, for example, ethyl alcohol, for addition to the aqueous cell suspension, while the various other extracting agents can be dissolved in water for such addition. The pH of the solution can be adjusted bythe addition of, for example, small amounts of sodium carbonate or sodium bicarbonate.

Turning now to the second step of the present method, the antigens extracted in the first step are separated from the remaining Whole cells and cell walls by any conventional separation method, for example, by centrifugation or by filtration. Centrifugation is the preferred method of separation. Ordinarily the suspension resulting from the first step herein is centrifuged for a period of time ranging from about 15 minutes to about 60 minutes at 5,000 to about 20,000 times the force of gravity. After centrifugation, the supernatant containing the extracted cell-free antigens is recovered. The centrifugate containing the cell walls and remaining whole cells is disposed to waste.

The extracted cell-free antigens recovered as an aqueous suspension can be used immediately or can be preserved with conventional stabilizing agents such as, for example, formalin or merthiolate. These suspensions can also be dried, for example, by freeze drying, to produce a dried product which can be conveniently stored for later use. This freeze drying can be carried out in a conventional freeze drying apparatus at a plate temperature ranging from about 15 C. to about 40 C. at a pressure of 1 mm. Hg or less. These suspensions can also be digested with trypsin before drying to improve the solubility of the dried product. This digestion is preferably carried out for from about 1 to about 3 hours at pH of about 8 and at a temperature ranging from about 35 C. to about 40 C., especially 37 C.; the concentration of trypsin present during digestion ranges from about 0.01 mg./ ml. to about 0.1 mg./ml.

The following examples are illustrative of the present invention and are not to be construed in any way as limiting the scope of the invention.

In these examples double diffusion precipitin tests for antigens and specific antibodies are carriedout as follows: An aluminum template is suspended over a 90 mm. glass Petri dish. This template comprises a central cylinder and six surrounding cylinders. Each of these cylinders is 6.5 mm. in diameter and 25 mm. long. The centers of the surrounding cylinders are each 14 mm. from the center of the central cylinder and are equally spaced about the circle which can be drawn through them. Each of the cylinders is suspended 1 mm. from the bottom of the Petri dish. Molten agar solution is poured into the Petri dish to partially fill the space beneath the template and to cover 2 mm. of the length of each of the cylinders. This molten agar solution is made up by mixing 0.6% highly purified agar (Ionagar), 0.01% merthiolate, and the balance 0.85% NaCl in water. The agar in the Petri dish is allowed to harden forming an agar plate. Then the template is removed exposing the solidified agar plate having molded therein seven cylindrical wells, each 2 mm. deep, formed by the seven cylinders of the template. Antiserum is added to the central well and extracted antigen to the outer wells, by use of Pasteur pipettes. The plate is then incubated at room temperature. During this incubation, antiserum and extracted antigen are added as needed to replenish the contents of the respective wells. The antiserum diffuses outward and is met by antigens diffusing inward whereupon lines or bands of antigenantibody precipitate form if specific antibody and antigen are present. Reactions are ordinarily visible in less than one week.

EXAMPLE I Cells of S. typhosa 0901 were harvested from ten 150 mm. plates of brain heart infusion agar, washed with 200 ml. water, and suspended in 200 ml. water in a beaker. The concentration of S. typhosa cells in the water suspension was approximately 5 mg./ml. S. typhosa 0901 cells are known to contain as their principal somatic antigens, the so-called O antigens.

To the suspension in the beaker was added with slight stirring 2.5 ml. of a 2% solution of dimethyldodecylamine in ethyl alcohol to provide a concentration of dimethyldodecylamine in the cell suspension of 250 p.p.m. The weight ratio of cells to extracting agent (di-methyldodecylamine) was 20:1. The cells were washed with the dimethyldodecylamine for minutes at 37 C. at a pH of 6-8. The suspension was then centrifuged for 30 minutes at 10,000 g to separate extracted 0 antigen from the remaining whole cells and cell walls.

The supernatant containing the extracted antigen was decanted. This decanted supernatant amounted to 200 ml. and contained substantially cell-free antigenic material amounting to about mg. To the supernatant, of formalin to preserve the extracted substantially cell-free somatic antigens which it contained.

Immune serum containing 0 antibody and specific for 0 antigen was then made up as follows: A 24-hour growth of S. typhosa 0901 on brain heart infusion agar was suspended in saline, heated for two hours at 100 C. suspended in 95% ethyl alcohol, washed twice in acetone and dried in vacuo (Roschkas method, described starting at page of Edwards, P. R. and Ewing, W. H., Identification of Enterobacteriaceae, Burgess Publishing Co., Minneapolis (1962). This preparation was administered in saline in five doses of 0.4 mg., 0.8 mg., 1.6 mg., 3.2 mg. and 6.4 mg. by intravenous injection in rabbits over a period of two weeks. The animals were bled 6 days after the last injection, and the serum was preserved with an equal volume of glycerol and refrigerated.

Double diffusion precipitin tests carried out as described above between the centrifuged supernatant and the above prepared immune serum gave an antigen precipitate indicating the presence of 0 antibody in the immune serum.

In another case, supernatant was prepared as above amounting to 200 ml. To this were added 10 mg. trypsin and 22 ml. 0.1 M tris(hydroxymethyl)amino methane buffer (pH 8.0). The mixture was incubated for two hours at 37 C. The resulting mixture was freeze dried in conventional apparatus to provide dry antigen extract. The dry extract was readily soluble in water. When dissolved in water the extract gives antigen-antibody precipitate when substituted for the supernatant in the above double diffusion precipitin test showing the presence of 0 antibody in the immune serum.

Similar extraction, separation, and precipitin test results were achieved to those above when 250 p.p.m., respectively, of dodecyltrimethylammonium bromide, Roccal, or disodium ethylenediaminetetraacetate were substituted for the 250 p.p.m. of dimethyldodecylamine above. These extracting agents were added to cell suspensions as 2% water solutions.

EXAMPLE l1 Extraction, separation, and precipitin test results similar to those achieved in Example I were achieved when equal concentrations of cells of S. typhosa Ty 2V were substituted for the cells of S. typhosa 0901 extracted in that example and otherwise the extraction, separation, and precipitin test conditions were the same as in EX- ample I.

In addition, double diffusion precipitin tests were carried out as generally described above between the supernatants recovered after the separation steps herein and immune serum specific for Vi antigen made with Citrobacter freundii (Ballerup 107) by Peluffos method (described starting at page 111 of Edwards, P. R. and Ewing, W. H., Identification of Enterobacteriaceae, Burgess Publishing Co., Minneapolis (1962). These tests gave antibody-antigen precipitates showing the presence of Vi antibodies in the immune serum.

The test results are those expected since S. typhosa Ty 2V is known to contain both 0 and Vi antigens as somatic antigens.

EXAMPLE III Cells of E. coli (ATCC 10536) were harvested from a 1 l. Penassay broth culture incubated for 24 hours at 37 C., washed twice with 200 ml. water each time, and then suspended in 400 ml. water in a beaker. The concentration of the E. coli cells in the water suspension was about 4 mg./ml.

To the suspension in the beaker was added with slight stirring 1.0 ml. of a 2% solution of dimethyldodecylamine in ethyl alcohol to provide a concentration of dimethyldodecylamine in the aqueous cell suspension of 50 p.p.m.

The weight ratio of cells to dimethyldodecylamine was 80:1. The cells were washed with the dimethyldodecylamine for 10 minutes at 37 C. and at a pH of 6-8. The suspension was then centrifuged for 30 minutes at 10,000 g to separate extracted antigens from the remaining whole cells and cell walls. The supernatant containing the extracted antigen was decanted and amounted to 400 ml. The decanted supernatant contained substantially cell-free somatic antigenic material amounting to about 128 mg.

Immune serum was made up as follows: Twenty-four hour Penassay broth cultures of E. coli (ATCC 10536) were heated at 100 C. for two hours and preserved with 0.3% formalin. Rabbits were given four intravenous injections of 0.5, 1, 2, and 3 ml. of this preparation at 5-day intervals, and bled five days after the last injection. The serum was preserved by the addition of 0.1% of its own weight of merthiolate.

Double diffusion precipitin tests were carried out as described hereinbefore between the decanted supernatant and the E. coli immune serum. These tests gave an antibody-antigen precipitate showing that the antigens contained in the supernatant can be used to detect E. coli antibodies in blood serum. The cells remaining in the centrifugate were much less agglutinable than untreated whole cells, indicating that the surface antigens in the treated cells had been removed.

Similar extraction, separation, precipitation and agglutination results to those achieved in Example III were achieved when 50 p.p.m., respectively, of dodecyltrimethylammonium bromide, or Roccal, were substituted for the 50 p.p.m. of dodecyldimethylamine in Example III. These extracting agents were added to cell suspensions as 2% Water solutions.

Extraction, separation and precipitin test results similar to those achieved in the above Examples I-III are also achieved when equal concentrations of other extracting agents, for example, decylhydroxymethylpropylamine,

tetradecyltrimethylammonium chloride, hexadecyltrimeth- 1 ylammonium iodide, dodecyldimethylbenzyl ammonium bromide, dimethyltetradecylbenzyl ammonium bromide, chloromethyldiethyltetradecylammonium chloride, dodecyltriethanolammonium methosulfate, tetradecylmethyldiethanolammonium ethosulfate, N-dodecylpiperidine, cetyl pyridinium chloride, N-dodecyl-1,3-propylene diamine, dimethyldodecylsulfonium iodide, dimethyldodecylsulfoxonium methosulfate, l-dodecyl-Z-imino imidazolidine, polymerized epichlorohydrin (5 moles of monomer per molecule) quaternized with three moles of dimethyldodecylamine or 1:1 by weight mixtures of DTAB and Roccal are substituted for the extracting agents in the above examples.

Cell-free somatic antigens prepared by the method of this invention are also useful in other ways. For example, it is believed that cell-free antigens of S. typhosa can be employed to produce vaccines which are more stable, more easily standardized, and less toxic than presently available vaccines comprised of suspensions of whole cells.

What is claimed is:

1. A method of preparing substantially cell-free somatic antigens comprising the steps of (1) washing intact E. coli cells in an aqueous solution of an extracting agent selected from the group consisting of:

(a) amines having the formula from 0 to about 1 amine group having the formula 8 and from 0*to about'2 halogen atoms and wherein each'R group is selected from the group consisting .of hydrogen, alkyl groups containing from 1 to about 3 carbon atoms and hydroxy alkyl groups containing from lto about 3 carbon atoms; (b) quaternary ammonium compounds having the formula wherein R has the definition hereinbefore given and each R group is selected from the group consisting of alkyl groups containing from 1 to about 3 carbon atoms, mono halogen substituted alkyl groups containing from 1 to about 3 carbon atoms, benzyl groups and hydroxy alkyl groups containing from 1 to about 3 carbon atoms, and wherein X is selected from the group consisting of iodide, bromide, methosulfate, ethosulfate and chloride anions;

(c) N-alkyl piperidines wherein the alkyl group contains from about 8 to about 18 carbon atoms;

(d) alkyl pyridinium halides wherein the alkyl group contains from about 8 to about 18 carbon atoms and the halide is selected from the group consisting of bromide, chloride and iodide;

(e) N-alkyl alkylene diamines wherein the alkyl group contains from about 8 to about 18 carbon atoms and the alkylene group contains from about 2 to about 4 carbon atoms;

(f) sulfonium and sulfoxonium compounds having the formula R4 R -iriru X wherein R is a hydrocarbon group containing from about 8 to about 18 carbon atoms, wherein each R group is an alkyl group containing from 1 to about 3 carbon atoms, wherein X has the definition hereinbefore given (e.g., selected from the group consisting of iodide, bromide, chloride, methosulfate and ethosulfate anions) and Y is selected from the group consisting of V,

8 and groups;

(g) vl-allryl-Z-imino imidazolidines wherein the alkyl group contains from about 8 to about 18 carbon atoms;

(h) polyquaternary ammonium compounds having the wherein R is a hydrocarbon group containing from 1 to about 24 carbon atoms, wherein each R group is a hydrocarbon group containing from 1 to about 4 carbon atoms, wherein X is selected from the group consisting of chlorine, iodine and bromine atoms, where L, m, and n are integers such that L is an integer from 0 to about 50, the sum of m and n is from about 2 to about 50, and the sum of m, n and the number of carbon atoms in R is greater than 12;

and

(i) mixtures thereof;

the concentration of said cells ranging from about 5 micrograms/ml. to about milligrams/ml; the concentration of said extracting agent ranging from about 1 ppm. to about 1000 ppm; the weight ratio of cells to extracting agent ranging from about 5:1 to about 100:1; while maintaining a temperature ranging from about 5 C.

to about 150 C. and a pH ranging from about 4 to about 12; and (2) separating the antigens extracted in the first step from the remaining whole cells and cell walls.

2. The method of claim 1 wherein the cell concentration ranges from about 1 milligram/ ml. to about 20 milligrams/ml, the extracting agent concentration ranges from about 25 p.p.m. to about 500 p.p.m., the weight ratio of cells to extracting agent ranges from about :1 to about 10011, the temperature ranges from about 35 C. to about 45 C., the pH ranges from about 6 to about 9', and the time of washing ranges from about 30 seconds to about 5 hours.

3. The method of claim 2 wherein the extracting agent is dimethyldodecylamine.

4. The method of claim 2 wherein the extracting agent is dodecyltrimethylammonium bromide.

5. The emthod of claim 2 wherein the extracting agent is C to C alkyldimethylbenzyl ammonium chloride.

6. A method of preparing substantially cell-free somatic antigens comprising the steps of (1) washing intact S. typhosa cells in an aqueous solution of an extracting agent selected from the group consisting of:

(a) amines having the formula wherein R is an alkyl group containing from about 8 to about 18 carbon atoms and having as substituents from 0 to about 1 amine group having the formula R! I -N and from 0 to about 2 halogen atoms and wherein each R group is selected from the group consisting of hydrogen, alkyl groups containing from 1 to about 3 carbon atoms and hydroxyl alkyl groups containing from 1 to about 3 carbon atoms;

(b) quaternary ammonium compounds having the formula [Rab -R 1 X wherein R has the definition hereinbefore given and each R group is selected from the group consisting of alkyl groups containing from 1 to about 3 carbon atoms, mono halogen substituted alkyl groups containing from 1 to about 3 carbon atoms, benzyl groups and hydroxy alkyl groups containing from 1 to about 3 carbon atoms, and wherein X is selected from the group consisting of iodide, bromide, methosulfate, ethosulfate and chloride anions;

(c) N-alkyl piperidines wherein the alkyl group contains from about 8 to about 18 carbon atoms;

(d) alkyl pyridinium halides wherein the alkyl group contains from about 8 to about 18 carbon atoms and the halide is selected from the group consisting of bromide, chloride and iodide;

(e) N-alkyl alkylene diamines wherein the alkyl group contains from about 8 to about 18 carbon atoms and the alkylene group contains from about 2 to about 4 carbon atoms;

(f) sulfonium and sulfoxonium compounds having the formula 34 ra r941 x wherein R is a hydrocarbon group containing from about 8 to about 18 carbon atoms, wherein each R group is an alkyl group containing from 1 to about 3 carbon atoms, wherein X has the definition hereinbefore given (e.g., selected from the group consisting of iodide, bromide, chloride, methosulfate and ethosulfate anions) and Y is selected from the group consisting of groups; (g) 1-alkyl-2-imino imidazolidines wherein the alkyl group contains from about 8 to about 18 carbon wherein R is a hydrocarbon group containing from 1 to about 24 carbon atoms, wherein each R group is a hydrocarbon group containing from 1 to about 4 carbon atoms, wherein X is selected from the group consisting of chlorine, iodine and bromine atoms, wherein L, m, and n are integers such that L is an integer from 0 to about 50, the sum of m and n is from about 2 to about 50, and the sum m, n and the number of carbon atoms in R is greater than 12;

(i) sodium and potassium salts of ethylenediamine tetraacetic acid; and

(j) mixtures thereof;

the concentration of said cells ranging from about 5 micrograms/ml. to about milligrams/mL; the concentration of said extracting agent ranging from about 1 p.p.m. to about 1000 p.p.m.; the weight ratio of cells to extracting agent ranging from about 5:1 to about 100:1; while maintaining a temperature ranging from about 5 C. to about C. and a pH ranging from about 4 to about 12; and (2) separating the antigens extracted in the first step from the remaining whole cells and cell walls.

7. The method of claim 6 wherein the cell concentration ranges from about 1 milligram/ml. to about 20 milligrams/ml, the extracting agent concentration ranges from about 25 p.p.m. to about 500 p.p.m., the weight ratio of cells to extracting agent ranges from about 5:1 to about 100:1, the temperature ranges from about 35 C. to about 45 C., the pH ranges from about 6 to about 9, and the time of washing ranges from about 30 seconds to about 5 hours.

8. The method or" claim 7 wherein the extracting agent is dimethy-ldodecylamine.

9. The method of claim 7 wherein the extracting agent is dodecyltrimethylammonium bromide.

10. The method of claim 7 wherein the extracting agent is C to C alkyldimethylbenzyl ammonium chloride.

11. The method of claim 7 wherein the extracting agent is the disodium salt of ethylenediaminetetraacetic acid.

No references cited.

RICHARD L. HUFF, Primary Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,408,440 October 29, 1968 Jack G. Voss It is certified that error appears in the above identified patent and that said Letters Patent are hereby corrected as shown below:

Column 2, lines 48 to 53, and column 8, lines 54 to 58, the formulas, each occurrence, should read as shown below:

HO CH CH -O- -CH -(|3H-O- -CH -CH-O H 1 CH CH X R -NR -X L R 111 n Column 3, line 25, "R should read R Column 6, line 5, after "natant" insert was added 0.3%, by weight of the supernatant line 23, after "antigen" insert antibody Column 9, line 17, "emthod" should read method Signed and sealed this 10th day of March 1970.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. WILLIAM E. SCHUYLER, JR.

Attesting Officer Commissioner of Patents 

1. A METHOD OF PREPARING SUBSTANTIALLY CELL-FREE SOMATIC ANTIGENS COMPRISING THE STEPS OF (1) WASHING INTACT E. COLI CELLS IN AN AQUEOUS SOLUTION OF AN EXTRACTING AGENT SELECTED FROM THE GROUP CONSISTING OF: (A) AMINES HAVING THE FORMULA
 6. A METHOD OF PREPARING SUBSTANTIALLY CELL-FREE SOMATIC ANTIGENS COMPRISING THE STEPS OF (1) WASHING INTACT S. TYPHOSA CELLS IN AN AQUEOUS SOLUTION OF AN EXTRACTING AGENT SELECTED FROM THE GROUP CONSISTING OF: (A) AMINES HAVING THE FORMULA 